Electrophysiological characterization of a recombinant human Na+‐coupled nucleoside transporter (hCNT1) produced in Xenopus oocytes

Open Access
Publisher Website
Google Scholar
Abstract
Human concentrative nucleoside transporter 1 (hCNT1) mediates active transport of nucleosides and anticancer and antiviral nucleoside drugs across cell membranes by coupling influx to the movement of Na+ down its electrochemical gradient. The two‐microelectrode voltage‐clamp technique was used to measure steady‐state and presteady‐state currents of recombinant hCNT1 produced in Xenopus oocytes. Transport was electrogenic, phloridzin sensitive and specific for pyrimidine nucleosides and adenosine. Nucleoside analogues that induced inwardly directed Na+ currents included the anticancer drugs 5‐fluorouridine, 5‐fluoro‐2′‐deoxyuridine, cladribine and cytarabine, the antiviral drugs zidovudine and zalcitabine, and the novel thymidine mimics 1‐(2‐deoxy‐β‐d‐ribofuranosyl)‐2,4‐difluoro‐5‐methylbenzene and 1‐(2‐deoxy‐β‐d‐ribofuranosyl)‐2,4‐difluoro‐5‐iodobenzene. Apparent Km values for 5‐fluorouridine, 5‐fluoro‐2′‐deoxyuridine and zidovudine were 18, 15 and 450 μm, respectively. hCNT1 was Na+ specific, and the kinetics of steady‐state uridine‐evoked Na+ currents were consistent with an ordered simultaneous transport model in which Na+ binds first followed by uridine. Membrane potential influenced both ion binding and carrier translocation. The Na+–nucleoside coupling stoichiometry, determined directly by comparing the uridine‐induced inward charge movement to [14C]uridine uptake was 1: 1. hCNT1 presteady‐state currents were used to determine the fraction of the membrane field sensed by Na+ (61%), the valency of the movable charge (−0.81) and the average number of transporters present in the oocyte plasma membrane (6.8 × 1010 per cell). The hCNT1 turnover rate at −50 mV was 9.6 molecules of uridine transported per second.